Functional Characterization of the
Non-Coding Control Region of Human Polyomaviruses
Inauguraldissertation zur
Erlangung der Würde eines Doktors der Philosophie vorgelegt der
Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel
von
Elvis Tasih Ajuh aus Kamerun
Basel, 2017
Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch
2 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von
Prof. Dr. Marcel Tanner
Prof. Dr. med. Hans H. Hirsch Prof. Dr. Volker Thiel
Basel, den 19.09.2017
Prof. Dr. Martin Spiess Dekan
3
TABLE OF CONTENTS
1 ABREVIATION ... 5
2 ABSTRACT ... 7
3 INTRODUCTION ... 9
3.1 Polyomaviridae ... 9
3.1.1 Structure and genome architecture of polyomavirus ... 11
3.1.1.1 Non-coding control region ... 12
3.1.1.2 Early viral gene region ... 13
3.1.1.3 Late viral gene region ... 13
3.1.2 Polyomavirus life cycle ... 14
3.1.2.1 Host cell receptors, viral entry and un-coating ... 14
3.1.2.2 Early viral gene expression ... 14
3.1.2.2.1 Large T antigen: the multifunctional protein ... 15
3.1.2.2.2 Viral encoded microRNA ... 16
3.1.2.3 Viral genome replication ... 17
3.1.2.4 Late viral gene expression ... 17
3.1.2.5 Encapsidation and viral release ... 18
3.1.3 The emerging family of polyomaviruses ... 19
3.1.3.1 Murine polyomavirus ... 19
3.1.3.2 Simian Virus 40 ... 20
3.1.3.3 Human Polyomaviruses ... 20
3.1.3.3.1 BK and JC polyomavirus ... 20
3.1.3.3.2 KI and WU polyomavirus ... 21
3.1.3.3.3 Merkel cell polyomavirus ... 22
3.1.3.3.4 Human polyomavirus 6 and 7 ... 23
3.1.3.3.5 Trichodysplasia spinulosa polyomavirus ... 23
3.1.3.3.6 Human polyomavirus 9 ... 24
3.1.3.3.7 Human polyomavirus 10 ... 24
3.1.3.3.8 St Louis polyomavirus ... 25
3.1.3.3.9 Human polyomavirus 12 ... 25
3.1.3.3.10 New Jersey polyomavirus ... 25
3.1.3.3.11 Lyon IARC polyomavirus ... 26
4
3.1.4 Human polyomaviruses and associated diseases ... 29
3.1.4.1 Human polyomaviruses associated non-cancerous diseases ... 31
3.1.4.1.1 BKPyV-associated diseases ... 31
3.1.4.1.2 TSPyV-associated disease ... 37
3.1.4.2 Human polyomaviruses and cancers ... 39
3.1.4.2.1 MCPyV-associated Merkel cell carcinoma ... 40
3.1.5 Systems for HPyV propagation and infection ... 44
4 AIMS OF THE THESIS ... 48
4.1 Hypotheses ... 49
4.2 Rationales ... 53
5 RESULTS ... 54
5.1 Designing a smaller bi-directional reporter vector lacking some sequences compared to the previous pHRG reporter vector ... 54
5.2 Novel human polyomavirus non-coding control regions differ in bi-directional gene expression according to host cell, large T antigen expression and clinically occurring rearrangements ... 61
5.3 Point mutations in the large T antigen binding sites of BK polyomavirus non- coding control region affect gene expression ... 103
5.4 Imperfect symmetry of Sp1 and core promoter elements regulates early and late viral gene expression of the bi-directional BK polyomavirus non-coding control region ... 124
5.5 Donor-derived urothelial cancer after kidney transplantation associated with a BK polyomavirus with increased oncogenic potential ... 146
6 DISCUSSION ... 174
7 SUPPLEMENTARY MATERIAL AND METHODS ... 178
8 REFERENCES ... 185
9 GLOSSARY ... 215
10 ACKNOWLEDGEMENTS ... 219
11 CURRICULUM VITAE ... 220
5 1 ABREVIATION
5HT2AR 5-hydroxy-tryptamine-2A serotonin receptor BKPyV BK polyomavirus
BKPyVAN BKPyV-associated nephropathy
BKPyVHC BKPyV-associated hemorrhagic cystitis
BP Base pairs
BRE B recognition element CMV Cytomegalovirus CNS Central nervous system CPE Core promoter element CSF Cerebrospinal fluid DNA Deoxyribonucleic acid
DPE Downstream promoter element
Ds-DNA Double-stranded deoxyribonucleic acid ER Endoplasmic reticulum
ERAD Endoplasmic reticulum-associated degredation EVGR Early viral gene region
HIV-1 Human immunodeficiency virus-1 HPyV10 Human polyomavirus 10
HPyV12 Human polyomavirus 12 HPyV6 Human polyomavirus 6 HPyV7 Human polyomavirus 7 HPyV9 Human polyomavirus 9 HPyVs Human polyomaviruses Inr Initiation element JCPyV JC polyomavirus LTag Large tumor antigen LVGR Late viral gene region MCPyV Merkel cell polyomavirus
6 mRNA Messenger ribonucleic acid
miRNA Micro RNA
NCCR Non-coding control region NK Cells natural killer cells NLS Nuclear localization signal
PBMCs Peripheral blood mononuclear cells
PML Progressive multifocal leukoencephalopathy pRB Retinoblastoma protein
PyVAN Polyomavirus associated nephropathy
PyVHC Polyomavirus associated hemorrhagic cystitis PyVs polyomaviruses
RNA Ribonucleic acid
RCA Rolling circle amplification RPA Replication protein A
rr-NCCR Rearranged non-coding control region sTag Small T antigen
STLPyV Saint Louis polyomavirus SV40 Simian virus 40
TSS Transcription start site
TSPyV Trichodysplasia spinulosa polyomavirus VLP(s) Virus-like particle(s)
Vp1 Major capsid protein 1 Vp2 Minor capsid protein 2 Vp3 Minor capsid protein 3 ww Wild type or Archetype
7 2 ABSTRACT
With the advent of advanced molecular biology techniques, 13 human polyomaviruses (HPyVs) have been identified from different human body compartments. Generally, infection with HPyVs is harmless in immunocompetent people. However, some of these viruses are known to cause severe morbidities in the immunosuppressed. Five HPyVs, BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), Merkel cell polyomavirus (MCPyV), Trichodysplasia spinolosa polyomavirus (TSPyV) and HPyV7 are known to cause diseases. Although other HPyVs have been detected in diseased tissues or cancers, evidence of their involvement is lacking.
Furthermore, less is known about the cell tropism, replication in cell culture, and gene regulation of HPyVs. The non-coding control region (NCCR) of HPyVs functions as a bi-directional promoter/enhancer system, coordinating the respective steps of the viral replication cycle. Furthermore, it also determines viral persistence, host cell specificity, replication and virulence. Efficient replication of BKPyV and JCPyV have been demonstrated in cell cultures, but less is known with regards to the novel HPyVs. NCCR activity which can be used as an indicator of HPyV cell tropism and replication in cell culture is lacking for the newly discovered HPyVs. Despite similarities in the genome organization of HPyVs, the NCCRs of the HPyVs are different with respect to large T antigen binding sites, transcriptional factor binding sites (TFBS) and length. We hypothesized that the HPyV-NCCRs will display different activity in the same or different host cells. A bi-directional reporter vector recapitulating the HPyV genome with a red fluorescence protein (dsRed2) and a green fluorescence protein (EGFP) as markers of EVGR and LVGR, respectively, was designed. The reporter was used to analyze the NCCR activity of the 13 HPyVs in different cell lines originating from kidney, skin, lungs, cervix, brain and colon cancers. Our result demonstrated that the bi-directional HPyV-NCCR activity substantially differ in the same and different host cells. Indicating that the HPyV- NCCRs differentially sense and interpret the host cells’ transcription factors and different host cells’ transcription factors modulate the basal HPyV-NCCRs expression.
As previously reported for BKPyV and JCPyV, rearranged (rr)-NCCR variants of newly discovered HPyVs associated with diseases showed higher EVGR expression compared to their respective archetype, indicating the NCCR a major pathogenicity
8 determinant. Analyzing the HPyV-NCCRs’ activity in cell lines expressing T antigens (Tags) displayed activation of EVGR expression, suggesting that the bi-directional reporter recapitulates an essential NCCR response reported for the viral replication cycle. Furthermore, it indicates that cell lines expressing Tags of respective HPyVs can be used for the propagation of respective HPyVs. This data serves as a basis for understanding host and viral factors that may permit the identification of suitable cell culture systems and secondary host cell tropism for HPyVs. However, actual viral replication studies are needed to confirm replication of HPyV genomes in some of the cell lines tested. Furthermore, the role of the LTag-binding motif with regards to the regulation of EVGR and LVGR has been well-characterized in the monkey polyomavirus, simian virus 40, although very little is known with respect to the role of the LTag-binding motif for HPyVs. Given that the LTag recognition sites influence EVGR and LVGR expression and the LTag-binding sites organization of SV40 is similar to that of some HPyVs, such as BKPyV, we hypothesized that the role of the LTag-binding motif in the regulation of EVGR and LVGR expression in BKPyV is potentially similar to that of SV40. The EVGR and LVGR expression of BKPyV archetype (ww)-NCCR was compared with those of BKPyV ww-NCCR defective in one or more LTag-binding motifs in HEK293, HEK293T and HEK293TT cell lines, expressing none, small and large amounts of SV40 LTag, respectively. Our results indicated that abundant LTag decreased EVGR-expression by probably interacting with LTag binding site A, suggesting abundant LTag expression may down regulate EVGR expression, similar to SV40’s-EVGR autoregulation. Contrarily, the LVGR expression is proportionally increased in the presence of increasing amounts of LTag expression (HEK293T and HEK293TT cells), by LTag potentially interacting with binding sites A and D. These results may open avenues for designing new therapeutic strategies targeting this reduction of EVGR expression in the presence of abundant LTag expression. Actual viral replication studies are needed to verify whether these mutants are replicative competent or not.
Concluding, a basic approach was used to elucidate factors that may allow the replication of HPyVs in cell cultures, that will greatly enhance our understanding of the basic biology of these viruses. Furthermore, putative host cell tropism of HPyVs was suggested. The effect of LTag-binding sites with respect to BKPyV’s EVGR and LVGR expression, which could be extrapolated to other HPyVs was shown.
9 3 INTRODUCTION
3.1 Polyomaviridae
Polyomaviruses (PyVs) belonging to the family Polyomaviridae infect a wide range of vertebrates from birds to mammals. However, PyVs are limited in the species they can productively infect (1). The Polyomaviridae Study Group of the International Committee on Taxonomy of Viruses has recently revised the family Polyomaviridae by a sequence- and host-based rationale (1). Based on the observed distance between large T antigen (LTag) coding sequences, at least 73 species of the family Polyomaviridae were classified into four genera (Alpha-, Beta-, Gamma- and Delta- polyomavirus) (2). The Alphapolyomavirus and Betapolyomavirus genera contain polyomaviruses that infect a variety of mammals. The gammapolyomavirus genus contains avian polyomaviruses, while the genus Deltapolyomavirus contains only four human polyomaviruses (HPyVs). The observed distance between LTag-coding sequence of HPyVs is shown (Fig. 1). However, the closest neighbor of a HPyV may be from a PyV from animals. At least 73 PyVs have been identified so far, of which 13 are HPyVs detected in different human-body compartments (Fig. 2) (2).
Specifically BK polyomavirus (BKPyV) (3), JC polyomavirus (JCPyV) (4), Karolinska institute polyomavirus (KIPyV) (5), Washington university polyomavirus (WUPyV) (6), Merkel cell polyomavirus (MCPyV) (7), human polyomavirus 6 (HPyV6) (8), human polyomavirus 7 (HPyV7) (8), Trichodysplasia spinulosa polyomavirus (TSPyV) (9), human polyomavirus 9 (HPyV9) (10), human polyomavirus 10 (HPyV10) (11), St.
Louis polyomavirus (STLPyV) (12) human polyomavirus 12 (HPyV12) (13), New Jersey polyomavirus (NJPyV) (14). While this work was in progress, the International Agency for Research on Cancer (IARC) in Lyon described a new PyV (LIPyV), which now needs confirmation as a HPyV (15). HPyVs generally cause asymptomatic infections in early childhood, which can then reactivate upon immunosuppression causing severe diseases. Of all these HPyVs, only BKPyV, JCPyV, MCPyV, TSPyV and HPyV7 are known to cause diseases (Fig. 2). The seroprevalence of HPyVs is up to 99% in the general adult population (Table 1) (16-18), suggesting a low-level infection of these viruses in the general population.
10 Fig. 1: A neighbor joining phylogenetic tree without distance corrections based on observed distance between HPyVs large T antigen coding sequences. The phylogram was constructed after multiple sequence alignment of the large T antigen sequences of the HPyVs using clustal omega. Next to each branch the evolutionary distance is depicted. Based on the tree, HPyVs could be grouped under 3 genera (Alpha-, Beta- and Delta-polyomavirus). GenBank accession numbers, from which the large T antigen sequences are based are found in table 6.
Genus Alpha-polyomavirus Genus Beta-polyomavirus Genus Delta-polyomavirus
11 Fig. 2: The 13 HPyVs identified from different compartment of the human body including LIPyV which was discovered recently but needs confirmation as a HPyV (indicated with the arrows). The year of identification of each HPyV is depicted. Only 5 HPyVs highlighted in yellow can cause diseases. BKPyV and JCPyV are the two HPyVs that can be efficiently propagated in cell culture.
3.1.1 Structure and genome architecture of polyomavirus
Polyomaviruses are non-enveloped viruses with a supercoiled double-stranded circular DNA genome of about 5000 base pairs (bp). The genome is associated with cellular histones to form a minichromosome. It is encapsidated in an icosahedral- shaped viral capsid of approximately 45 nano meter (nm) in diameter (19, 20). The capsid is made up of 72 pentamers of the major capsid protein, viral protein 1 (Vp1), which is capable of self-assembly into viral-like particles (VLPs) (19, 21). Each Vp1 pentamer is associated with a minor capsid protein, Vp2 or Vp3 (Fig. 3A). The genome of polyomaviruses is divided into 3 main regions, the non-coding control
12 region (NCCR) and two transcriptional regions; early viral gene region (EVGR) and the late viral gene region (LVGR) (Fig. 3B). They are termed EVGR and LVGR due to the time of transcription during the course of the viral replication cycle. The EVGR and LVGR are transcribed in opposite strands and directions by the host cell RNA polymerase II as a single pre-messenger RNA.
3.1.1.1 Non-coding control region
The NCCR functions as a bi-directional promoter, harboring the origin of viral replication (ori), early and late promoters, enhancers regions, transcriptional start sites and a multitude of transcription factor-binding sites (TFBS). The NCCR can be referred to as the ‘’brain’’ of polyomaviruses. It is defined as the region between the start codon of the LVGR and the start codon of the EVGR. In BKPyV and JCPyV, the NCCR region proximal to the LVGR contains enhancer elements, LVGR transcriptional initiation sites and usually undergoes rearrangements including deletions, duplications and point mutations (22), whereas the region proximal to the EVGR harbors the origin of viral genome replication, EVGR transcriptional initiation sites and mostly not rearranged (23). This region also contains palindromes and dyad symmetry elements (23). Rearrangements of NCCR region proximal to the LVGR region result in different strains of the same species, which confers tissues specific expression of EVGR and LVGR expression. Rearranged BKPyV and JCPyV NCCR variants are common in BKPyV- and JCPyV-associated diseases (24-26).
Nonetheless, BKPyV-induced hemorrhagic cystitis and nephropathy was not linked to any particular NCCR architecture (27). Furthermore, polyomaviruses containing similar NCCR rearrangement have been reported in different patient groups and healthy individuals (18, 28). The exact role of rearranged NCCR variants in pathogenesis and the mechanisms of generating the rearrangements are poorly understood. Rearranged BKPyV and JCPyV NCCR variants with diverse duplications and deletions from patients, have been reported to increase EVGR expression, replication capacity and cytopathology in vitro (25, 26). This suggests that BKPyV and JCPyV variants with rearranged NCCR can be linked to increase EVGR expression, replication capacity and disease (22, 25), which has not been studied for the novel HPyVs. A multitude of TFBS within the HPyV NCCR control the cell specific EVGR and LVGR expression of HPyVs. The roles of some of these TFBS in the
13 control of BKPyV and JCPyV gene expression are known (29-34), whereas little or no information is known with respect to the newly discovered HPyVs. Nonetheless, since some of the TFBS are conserved among HPyVs, the knowledge gained from BKPyV and JCPyV can be extrapolated to the other HPyVs.
3.1.1.2 Early viral gene region
The EVGR pre-mRNA is spliced into different variants encoding for large T antigen (LTag), small T antigen (sTag) (Fig. 4) and other T antigens variants, which are produced by alternative splicing. Some of the HPyVs encode additional early proteins.
BKPyV encodes 17kT, JCPyV; T’135, T’136 and T’165, MCPyV; 57KT and ALTO, TSPyV; tinyT, 21kT, MTag and ALTO HPyV9; 145T, STLPyV may encode 229T, HPyV12; 84T and NJPyV may encode 299T (18).
3.1.1.3 Late viral gene region
The LVGR encodes the viral structural proteins, Vp1, Vp2, Vp3 and a non-structural late protein, agno, which are produced by alternative translational start sites. Only simian virus 40 (SV40), BKPyV and JCPyV encode the late non-structural protein, agno. The capsid of PyVs are composed of 72 pentamers of the major capsid protein Vp1, which are strengthened by disulphide and calcium bridges, and conserved throughout the polyomavirus family (20). These Vp1 pentamers form barrel-like structures, and within the hollows of Vp1, are contained minor capsid proteins, Vp2 and Vp3.
HPyV Genome
no (Ag ) NCCR
EVGR LVGR
VP1 VP3 VP2
sTag LTag
miRNA -1
miRNA -2 alt. splic
ed Tags
B A
14 Fig. 3: (A) Schematic representation of the HPyV viral particle. (B) Representation of general features of the HPyV genome.
3.1.2 Polyomavirus life cycle
3.1.2.1 Host cell receptors, viral entry and un-coating
The detailed mechanisms of polyomavirus entry, trafficking through the cytoplasm and nuclear entry is not well understood. However, much of the understanding of the polyomavirus life cycle (Fig. 5) have been deduced from investigating SV40, BKPyV and JCPyV infection mechanisms (35). Vp1 is known to interact with the host cell receptors, as for instance mutation of the Vp1 calcium binding residues block viral entry into the host cell, resulting to disruption of virion assembly (36). The monkey polyomavirus, SV40 binds the monosialotetrahexosylganglioside (GM1) receptor and enters the cell via caveolar-mediated endocytosis. Additionally, SV40 can also independently enter the cell via caveolin or clathrin mediated endocytosis (37). On the other hand, BKPyV uses the disialoganglioside GT1b and trisialogangliosides GD1 as receptors to enter the cells by caveolar-mediated endocytosis, while JCPyV binds to serotonin 5-hydroxytryptamine 2A (5-HT2A) and enters the cell via clathrin- mediated endocytosis (38, 39). After internalization, the viral particle is trafficked through the microtubule network to the endoplasmic reticulum (ER) (40-42). ER transiting is unique to PyVs, no other DNA virus known to replicate in the nucleus uses this route. Interactions between the PyVs capsid proteins and the ER proteins have been well studied in SV40 (43). Partial un-coating of the viral particle takes place in the ER. The disulfide-isomerase unlinks the disulfide bonds between the Vp1 pentamers in the capsid (40, 44). The minor capsid proteins Vp2 and Vp3 become accessible with the help of chaperone proteins, which is important for subsequent steps in the viral life cycle. Once the viral particle is partially disassembled, it is then retrograde-transported from the ER to the cytoplasm via the ER associated degradation (ERAD) pathway. The viral particle is then guided into the nucleus via the nuclear pore with the help of the nuclear localization signal (NLS) present in Vp2 and Vp3 (45).
3.1.2.2 Early viral gene expression
15 The early and late promoters within the NCCR control the bidirectional EVGR and LVGR transcription. The EVGR and LVGR transcription progresses outward from the NCCR via opposite strands of the genome. Once the virus gets into the nucleus, EVGR transcription is initiated by cellular RNA polymerase II and controlled by cis- acting sequences within the NCCR, such as TATA box, GC-rich sequences and enhancers. A single pre-EVGR mRNA is produced which undergoes alternative splicing into sTag, LTag and other T antigens spliced variants. The T antigens’
coding regions are overlapping at the N-terminal sequence, but differ at the C- terminal sequence (46).
3.1.2.2.1 Large T antigen: the multifunctional protein
The LTag of HPyVs is a multifunctional protein that interacts with numerous cellular and viral factors to initiate viral gene expression and viral genome replication (1). The structure and functions of the LTag have been largely described in the SV40 model, which is also relevant for BKPyV and JCPyV with amino acids homology of approximately 81% between SV40 and BKPyV, and 79% between SV40 and JCPyV (47). The large T antigen contained five regions that are conserved among HPyVs, that is, DnaJ region, LXCXE motif, nuclear localization signal, origin-binding domain (OBD) and ATPase/helicase domain. LTag binds to pentanucleotide sequences, GRGGC or its compliment GCCYC at the ori via the OBD region (48). The NCCR of all HPyVs contains at least one of these pentanucleotide sequences (49). Large T antigen binds to the ori within the NCCRs, unwinds the duplex DNA viral genome by using the ATPase/helicase activity (48). The ATPase gives the energy required for the helicase activity, while a zinc-binding domain proximal to the helicase domain is needed for the formation of LTag hexamer at the ori (48). In addition, LTag drives the cell into S-phase of the cell cycle resulting in the activation of DNA damage response (DDR) in order to promote replication of viral genome. The DnaJ domain cooperates with a proximally located motif called LXCXE to bind the retinoblastoma tumor suppressor proteins, pRB/p130/p107 (48). This binding disrupts the interactions between E2F (E2 transcription factor) and pRB/p130/p107. Dissociating pRB from E2F permits the formation of E2F-E2F dimers, which interacts with its target sequence and drives S-phase progression and DNA synthesis (48). Normally, improper entry of the host cell into S-phase would signal a p53-mediated apoptosis.
16 However, to circumvent the p53-mediated apoptosis, LTag binds p53 via the P53- binding domain within the helicase domain of LTag and allows progressive viral replication (1, 50). This p53 protein is a critical tumor suppressor in humans and its involvement in tumorigenesis has been widely demonstrated in the SV40 model (48, 51).
Likewise, sTag also has tumorigenic potentials. It promotes cells growth and viral replication by interacting with protein phosphatase 2A (PP2A), and disrupts its phosphatase ability (52). It has been reported to stimulate intercellular kinases to also promote cellular growth pathways (53).
Fig. 4: Schematic representation of the large and small T antigens.
3.1.2.2.2 Viral encoded microRNA
During the viral life cycle, small noncoding RNAs (miRNAs) post-translationally regulate gene expression by directed-mRNAs cleavage or repression of translation.
SV40, BKPyV, JCPyV, and MCPyV encode miRNA that target and deplete early viral transcripts, such as LTag mRNA (54-56). SV40 miRNA mutant infected cells were observed to be more sensitive to T antigen specific cytotoxic T-cell lysis in vitro, suggesting the importance of miRNA in viral evasion of the host immune response.
However, similar amounts of infectious virions were produced with the SV40 miRNA mutant compared to the wild-type (55). Moreover, the stress-induced ligand, UL16 binding protein 3 (ULBP3), which is important in the activation of the natural killer T- cell receptor (NKG2D), has been reported to be targeted by BKPyV and JCPyV
DnaJ LXCXE
p130 pRB p107
DnaJ
Unique region Zn finger
PP2A
NLS OBD Helicase p53
HSC70
HSC70
Large T-anHgens
Small T-anHgens
17 miRNA (57). The NKG2D mediated killing of infected cells was reduced due to the downregulation of ULBP3 by the viral miRNA. This indicated that PyVs could use this mechanism to latently infect the host without being eliminated by the host immune system (57). Interestingly, archetype (ww) BKPyV encoded miRNA has been reported to be involved in limiting BKPyV ww replication in renal tubular epithelia cells (58). Nevertheless, regulation of the miRNA could be accomplished through the balance of regulatory elements within the NCCRs controlling EVGR and miRNA expression prior to genome replication (58). The BKPyV and JCPyV miRNA is encoded on the late strand with complementarity to the 3′ coding end of the T antigen mRNA (Fig. 2B) (54). Since the miRNA is located on the late strand, one could suggest that the miRNA is expressed from the late promoter, although it may also be expressed from within or outside the NCCR. The miRNA of BKPyV and JCPyV are identical; this indicates that in the case of co-infection, the miRNA from each virus can downregulates each other’s T antigens expression. Since PyVs use miRNA to evade the host immune system, therapeutically targeting miRNA could be used for treating PyVs-associated diseases. Therefore, investigating the presence and function of miRNA of the other HPyVs is warranted.
3.1.2.3 Viral genome replication
PyVs depend on the host cell machineries to replicate its genome. LTag coordinates viral genome replication by recruiting multiple host factors required for viral genome replication. LTag monomers bind to the viral ori and assemble into two hexamers.
Once the binding to ori occurs, LTag uses its helicase activity to unwind the double stranded DNA (48) (as explained above). Next, replication protein A (RPA) is recruited to bind the single stranded unwound DNA, to prevent it from annealing and forming secondary structures.
3.1.2.4 Late viral gene expression
Concomitantly to viral genome replication, late viral proteins are expressed. LTag begins suppression of EVGR expression at this point, but the exact mechanism is not well understood. It is suggested that, high LTag amounts binding the ori, particularly to LTag binding site I in SV40 (59), results in the downregulation (auto-regulation) of EVGR expression and promotion of LVGR expression. Additionally, miRNA could
18 also be involved as explained above. LVGR transcription produces at least 3 structural proteins, Vp1, Vp2 and Vp3. They are encoded in an overlapping manner;
Vp3 is completely encoded in the 3’ end of Vp2. SV40 has been reported to encode Vp4 (60). Furthermore, unlike the other HPyVs the LVGR of SV40, BKPyV and JCPyV encodes a small late non-structural protein called agno (6, 7, 61, 62).
3.1.2.5 Encapsidation and viral release
In baculovirus expression systems, Vp1 can self-assemble into the capsid protein without the presence of Vp2 and Vp3, but Vp1 cannot assemble into the capsid protein in the cytoplasm, which might be due to low calcium concentrations in the cytoplasm. This theory was supported by the higher calcium concentration in the nucleus, where virions were observed by confocal and electron microscopy (19, 21, 63). The LVGR capsid proteins made in the cytoplasm are transported to the nucleus with the help of the nuclear localization signal sequence within the proteins (NLS).
Once in the nucleus they are assembled into virions. Vp1, the major capsid protein associates with minor capsid proteins Vp2 or Vp3 (not encoded by MCPyV), which extend internally anchoring the minichromosome. Viral release of the fully assembled infectious particles is poorly understood, but thought to occur via a number of processes including host cell lysis, agnoprotein increasing membrane permeability, Vp3 lytic properties, SV40 Vp4-mediated viroporin release (60, 64, 65). Vp4 was reported to be encoded by SV40 and function as a viroporin to form pores for viral release (60). However, recently, Henriksen et al. demonstrated that Vp4 is not important for viral release, since there wasn’t any difference between wild-type and Vp4 mutant release (66).
19 Fig. 5: Schematic representation of polyomavirus life cycle. 1. Attachment of host- cell receptors. 2. Entry by endocytosis. 3. Transport of viral particle via endosomes to ER and later to the nucleus via the nuclear pore. 4 and 5. EVGR transcription and translation. 6. Nuclear translocation of LTag and viral genome replication. 7. LVGR transcription and translation. 8. Newly-synthesized viral genomes encapsidation. 9.
Infectious viral particles release.
3.1.3 The emerging family of polyomaviruses
3.1.3.1 Murine polyomavirus
The mouse polyomavirus (MPyV) was one of the first PyV to be isolated by Ludwig Gross in 1953 while studying murine leukaemia virus (67). Heat inactivation of an inoculum, which usually protects against infection with Murine leukaemia virus, still resulted in leukaemia and tumors in the parotids glands of newborn mice. Thus, he concluded that the tumor was due to another infectious agent. Gross named this
20 infectious agent after further experimentation “polyoma” due to its ability to cause multiple tumors in various tissues in newborn mice. Since the MPyV discovery, viral oncogenesis has been intensively studied (46).
3.1.3.2 Simian Virus 40
SV40 was discovered in 1960 as a contaminant of a batch of polio vaccines generated in primary kidney cells from Rhesus monkeys (68). Between 1955 and 1963 approximately 100 million adults and children received the polio vaccine and the same methods for generating the vaccine were used throughout the world. A vacoulating agent, later named Simian virus (SV40) was identified during safety testing in some cultures causing cytopathic effect (1, 68). Furthermore, investigations of culture systems infected with SV40 revealed that this virus had the ability to cause tumors in animal models (69). SV40 has been linked to non-Hodgkin lymphoma, adult and pediatric brain tumors, mesothelioma, and osteosarcoma (70).
Epidemiological studies show that populations exposed to the SV40-contaminated polio vaccine failed to show increased risk of tumor development (71-73). However, the understanding of cell cycle and oncogenic pathways that permit the discovery of tumor suppressors pRB and p53 was greatly improved due to SV40 research (1, 46, 74). For more than 50 years the question of whether SV40 infection is an agent of human cancer has remained highly controversial (75).
3.1.3.3 Human Polyomaviruses 3.1.3.3.1 BK and JC polyomavirus
BKPyV and JCPyV were the two HPyVs discovered in 1971. They were named after the initials of the two patients from whom the viruses were isolated (3, 4, 76). BKPyV was isolated from the urine of a kidney transplant recipient (3), while JCPyV was isolated from the brain tissue of a progressive multifocal leukoencephalopathy (PML) patient (3, 4, 76). Inoculating human fetal brain tissue with isolated JCPyV agent revealed that JCPyV was the causative agent of PML, a fatal demyelinating disease in the central nervous system (CNS) (4). Infection of BKPyV and JCPyV occurs during early childhood. Seroprevalence of BKPyV and JCPyV in the general adult population ranges from 50-98% (77). The mechanism of transmission of BKPyV and
21 JCPyV is poorly understood, but thought to be via the respiratory or fecal-oral route (78-80). After primary infection of BKPyV or JCPyV, the viruses establish a persistent infection, possibly in kidney, neuronal, hematopoietic or lymphoid tissues (81-83).
BKPyV has been detected in various organs/compartments of the human body;
peripheral blood mononuclear cells (PBMCs), brain cells, urogenital cells, lymphocytes, cervix, rectum, sperm, skin and numerous cancers such as, neuroblastoma, osteosarcoma, prostate, cervix and Kaposi’s sarcoma (84-87).
JCPyV has been detected in PBMCs, kidney, lungs, bone marrow, gastrointestinal tract, tonsils, and lymphoid organs and in various cancers such as, B-cell lymphoma, lung, colon, urothelial, prostate, esophageal and cancers of the CNS (88-91).
Reactivation of JCPyV and BKPyV can occur upon immune suppression. The incidence of PML has increased greatly with the pandemic of acquired immunodeficiency syndrome (AIDS) in the 1980s (90). BKPyV reactivation occurs mostly in renal transplant recipients resulting in nephropathy, which is associated with the risk of organ rejection (90, 92-94). Reactivation of BKPyV in bone marrow transplant (BMT) recipients could lead to hemorrhagic cystitis (95, 96). Both viruses are known to have four main serological subtypes, however, the clinical importance of these subtypes is poorly understood (97)
3.1.3.3.2 KI and WU polyomavirus
Approximately 36 years after the discovery of the first two HPyVs, the interest in human polyomaviruses was rekindled in 2007, when two new HPyVs, KIPyV and WUPyV, were discovered (6, 62). Similar to the initial detection of BKPyV and JCPyV, KIPyV and WUPyV were reported within one month of each other. Using large-scale molecular virus screening, KIPyV was detected in a respiratory secretion at the Karolinska institute (KI) by Allander et al. Within a month, Gaynor et al., identified WUPyV in a respiratory secretion from a child with clinical signs of pneumonia at the Washington University (WU) by using high throughput molecular sequencing techniques (6, 62). Furthermore, these viruses were also detected in respiratory secretions of children with acute respiratory illness (98). Since then, they have also been detected in blood, faces, urine, lung, tonsils, cerebrospinal fluid, spleen, lymphoid tissue, brain of HIV-infected patients with and without PML (99-102).
Both viruses are phylogenetically related to each other and belong with BKPyV and
22 JCPyV to the Genus Betapolyomavirus (2, 6). The detection rates of both viruses in respiratory secretions by polymerase chain reaction (PCR) were between 0.4%-9%
in studies conducted throughout the world (5, 6, 62, 98, 103-116). Detection rates in immunocompromised populations are estimated to be higher, and many of the reports lack appropriate control population and used small cohorts. The detection of KIPyV and WUPyV with other respiratory pathogens is common, thus making it difficult to draw the etiological link between these two viruses with respiratory illnesses (6, 62, 116, 117). The seroprevalence of KIPyV and WUPyV in healthy adults’ ranges between 55%-93.3% and 54%-90%, respectively. This suggests a wide spread of KIPyV and WUPyV infection (77, 118, 119). Like BKPyV and JCPyV, it is hypothesized that KIPyV and WUPyV may persist in their host, reactivate and cause disease in immunosuppressed individuals. So far, no study has shown a significant difference between KIPyV and WUPyV detection rates in immunocompetent or immunocompromised individuals, although higher viral loads of KIPyV have been reported in the immunocompromised host (101, 120-127).
Currently, no possible link of KIPyV and WUPyV to cancer has been made. Moreover, a role for both viruses in different cancer types is possible (128-130). Lastly, there have been no definitive association of these viruses with any disease.
3.1.3.3.3 Merkel cell polyomavirus
One year later (2008), Merkel cell polyomavirus (MCPyV) was detected using a digital transcriptome subtraction technique of RNA extracted from a Merkel cell carcinoma (MCC) (7). MCC is rare and aggressive skin cancer predominantly found in elderly and immunocompromised individuals. 50% of individuals with advanced cases live at least 9 months after being diagnosed with MCC, which is greatly reduced in the immunocompromised (7, 131). Compared to SV40, the genome of MCPyV has higher sequence homology to Lymphotrophic polyomavirus (LPyV), a polyomavirus isolated in 1979 from a B-lymphoblastoid African green monkey cell line. DNA of MCPyV has been detected in the peripheral blood of both healthy and immunosuppressed individuals and many other human samples/organs such as:
eyebrow hairs, gall bladder, oral samples, lung, tonsils, intestine, appendix, liver, blood, saliva, lymphoid tissue, urine, malignant and non-malignant tonsillar tissues (132-137). Unlike other HPyVs, MCPyV is more closely related to MPyV.
23 Nonetheless, T antigens of MCPyV have the conserved features of the other HPyVs including; DnaJ binding domain, OBD, helicase/ATPase domain and conserved pRB binding motif (7). The seroprevalence of MCPyV in the healthy adult population ranges between 25%-42% (77). MCPyV was initially found integrated into the human genome (7). Viral integration into the host genome has also been reported for other polyomaviruses that cause tumor in cell culture models, but not before the discovery of MCPyV (7).
3.1.3.3.4 Human polyomavirus 6 and 7
Two new HPyVs were identified on the skin of adults in 2010 (8). HPyV6 and HPyV7 were detected by rolling circle amplification (RCA) from forehead swabs of volunteers (8). Small amounts of HPyV6 and HPyV7 virions are shed from skin of infected individuals. Some initial investigations detected 5/35 people to be positive for HPyV6 and 4/35 positive for HPyV7. These viruses have been detected in cervical specimens of HIV infected and non-infected, malignant and non-malignant tonsillar tissues, respiratory samples, feces and urine of an allogeneic stem cell transplant recipient. LTag of HPyV7 has been found in 23/37 thymomas (62.2%) and 8/20 thymic hyperplasia (40%) tissues. Furthermore, HPyV7 have been detected in PBMCs and skin of single and double lung transplant recipients with pruritic rash (133, 138-140). In addition, both viruses were detected in immunosuppressed patients with pruritic and dyskeratotic dermatoses (141). This suggests that these viruses can cause disease in immunocompromised patients. Seroprevalence studies using VLPs revealed that, HPyV6 and HPyV7 infect 69% and 35% adults, respectively, indicating that exposure to these viruses is common (8). So far definitive association of these viruses to tumorigenesis is unknown (142).
3.1.3.3.5 Trichodysplasia spinulosa polyomavirus
In 2010, another human polyomavirus called TSPyV was detected on the skin by RCA (9). TSPyV is the causative agent of Trichodysplasia spinulosa (TS) (9). TS is a rare skin disease affecting mostly immunocompromised persons (9, 143). The virus together with HPyV9, HPyV12, MCPyV, NJPyV and LIPyV belong to the genus Alphapolyomavirus (Fig. 1) (2). The clinical development of TS occurs as an eruption of erythematous papules with many follicular extensions localized in the limbs and
24 central face, mostly the nose (144), which was initially reported by Izakovic et al from a 31-year-old kidney transplant recipients undergoing treatment with cyclosporine A (145). Using quantified PCR and electron microscopy (EM) demonstrated that TSPyV was actively infecting TS follicular biopsy (144, 146). It has also been detected in 4%
of skin swabs from kidney transplant recipients without clinical TS, indicating that this virus could be found on the skin without causing disease (9). In addition, this virus has been detected in renal allograft of a kidney transplant patient, heart, spleen, lung, colon, bronchus, small intestine, liver, respiratory samples, feces, tissue from a patient with myocarditis and in tonsillar tissues of healthy individuals (138, 147-149).
TSPyV has high seroprevalence of about 70% in adult populations (150). This supports the theory that infection with this virus is usually asymptomatic causing TS only in highly immunocompromised individuals (150).
3.1.3.3.6 Human polyomavirus 9
In 2011, HPyV9 was detected in the serum of a kidney transplant recipient using degenerate primers (10). Whole genome sequencing of HPyV 9 revealed it to be closely related to LPyV and contained all the typical genomic characteristics of a polyomavirus (10). LPyV has been detected in blood in both immunosuppressed and healthy people and has a seroprevalence of 30% (77, 151, 152). A seroprevalence of 40% was determined for HPyV9, although cross reactivity of LPyV and HPyV9 was confirmed (153). This cross reactivity may account for the previous serological evidence of LPyV in human infection (153, 154). HPyV9 has been detected on skin swabs of MCC patients. This is in accordance with the finding that in MCC patient groups, HPyV9 was more common compared to age-matched controls (154).
Furthermore, HPyV9 variant was detected in PBMCs of an AIDS patient (155).
3.1.3.3.7 Human polyomavirus 10
HPyV10 was discovered in 2012 in chondyloma specimens from a patient with warts, hypogammaglobinemia, infections, and myelokathexis-congenital disorder of leukocytes (WHIM) syndrome (11, 156). In the same, year a strain of HPyV10 termed Malawi polyomavirus (MWPyV) was detected in stool samples of healthy children from Malawi using shot gun 454 pyrosequencing (11). Another nearly genetically identical strain called Mexican polyomavirus (MXPyV) was detected in stool samples
25 of Mexican children (157). MWPyV strain was also found in one respiratory sample and stool samples from healthy Mexican children. Furthermore, MWPyV was found in tonsils and adenoids of healthy children, whereas HPyV10 was also detected on skin of HIV-infected and non-infected men suggesting skin tropism for HPyV10 (157, 158).
3.1.3.3.8 St Louis polyomavirus
Like MWPyV, STLPyV was detected in a stool sample from a healthy child in 2013 using 454 pyrosequencing (12). STLPyV is placed in the genus Deltapolyomavirus (2). The virus shows high sequence homology to HPyV6, HPyV7 and HPyV10 in the LTag coding sequence (2). STLPyV has been reported to encode a unique protein called 229T, obtained through alternative splicing of the EVGR pre-mRNA (18). Stool specimens collected from children confirmed the presence of STLPyV, however there was no statistical association between STLPyV and gastroenteritis. STLPyV has also been identified from a skin wart of a WHIM syndrome patient (12, 159). Compare to other HPyVs, STLPyV has a high seroprevalence of about 68%-77%, with age- stratified data indicating early childhood acquisition of this virus (160).
3.1.3.3.9 Human polyomavirus 12
HPyV12 was detected in resected human liver tissues and also in rectum, colon and a fecal sample (13). Studies in healthy adults/adolescents and pediatric children revealed a seroprevalence of 23% and 17%, respectively (13), indicating that primary infection of HPyV12 occurs during early childhood. So far, this virus has not been associated with any disease.
3.1.3.3.10 New Jersey polyomavirus
NPyV was detected in endothelial cells of a pancreatic transplant recipient suffering from necrotic plaques on multiple areas of the skin and retinal blindness (14). High viral load and viral particles of NJPyV within the nuclei of vascular endothelia cells were detected by specific real time PCR and EM (14). Reduction of immunosuppression resulted to slow subsiding of the patient’s weakness, but the patient’s eyesight failed to return. NJPyV viremia was detected until 10 months after the onset of symptoms, even though the patient showed improvement of symptoms.
Furthermore, it was not possible to detect NJPyV in sera collected from the general
26 population or in the patient’s pre-transplant serum (14).
3.1.3.3.11 Lyon IARC polyomavirus
The most recently LIPyV was discovered from the skin swabs, oral gargles and an eyebrow hair of cancer-free individuals by using a sensitive degenerative PCR and next-generation sequencing (15). The virus is phylogenetically related to the raccoon PyV identified in neuroglial tumors in free-ranging raccoons. The biological properties of this PyV is yet unknown.
27 Table 1: HPyVs discovery and potential and proven associated-diseases. N.A: not applicable.
Species/
common Name
Year discovered/
references
Initial biological compartment isolated/detected
another biological compartment detected
Associated-diseases Seroprevalence / references
BKPyV/
HPyV1 1971 (3)
Urine of kidney transplant recipient
Bladder, blood, bone, brain, CNS, heart lung, skin, spleen, genital, salivary glands
BKPyV associated- hemorrhagic cystitis (BKPyVHC) (96), BKPyV associated-nephropathy
BKPyVVAN) (161), progressive multifocal leukoencephalopathy (PML) (162), retinitis (162), pneumonitis (163) meningitis and encephalitis (164), ureteral stenosis (165), prostate cancer (166),
55–90%
(77, 167)
JCPyV/
HPyV2 1971 (4) CSF of a PML patient
Brain, CNS, kidney, lungs, GI tract, blood, tonsils
PML (4), colon cancer (168), multiple sclerosis (MS) (169)
44-90%
(77, 167)
KIPyV/
HPyV3 2007 (62) Respiratory tract secretions
Blood, stool, brain, tonsil, CNS, lung cancer tissue, lymphoid and tonsil tissues.
respiratory disease (18) 55-91%
(77, 118, 170)
WUPyV/
HPyV4 2007 (6) Respiratory tract secretions
Blood, stool, brain, tonsil, CNS, CSF, lymphoid and tonsil tissues.
respiratory disease (171) 69-98%
(77, 118, 170)
28
MCPyV/
HPyV5 2008 (7) Skin of Merkel cell carcinoma patients
Plucked eyebrow hairs, healthy skin, liver, urine, oral samples, tonsils, gall bladder, lung, saliva, oral samples
80% of Merkel cell carcinoma (7) 58-96%
(77, 170, 172- 174)
HPyV6
2010 (8) Human skin swab
Eyebrow hair, stool sample, urine,
nasopharyngeal swabs
BRAF inhibitor-induce epithelia proliferations, pruritic and
dyskeratotic dermatoses (48, 141)
67–98%
(8, 170, 172, 174, 175)
HPyV7
2010 (8) Human skin swab
Eyebrow hair, stool sample, urine,
nasopharyngeal swabs
Pruritic and dyskeratotic
dermatoses (48, 141), thymomas (139)
35–86% (8, 170, 172, 174, 175)
TSPyV/
HPyV8 2010 (9)
Skin of
Trichodysplasia spinulosa
immunocompromi se patient
Heart, lungs, liver, small intestine, colon, allograft of kidney transplant patient,
bronchus, colon tissue,
Trichodysplasia spinulosa (48) 70–84%
(150, 170, 172, 174)
HPyV9
2011(10)
Serum sample of kidney transplant recipient
Skin of Merkel cell carcinoma patient, Skin of healthy people
N.A 20–70% (153,
170, 172, 174- 176)
HPyV10
2012 (11, 157)
Stool sample (MW)/ Condyloma specimen (10w)
Forehead swap, stool, respiratory samples
Genital wart,
Hypogammaglobulinemia,
infections, Myelokathexis (WHIM) syndrome
(11, 156)
42–99%
(170, 177, 178)
STLPyV/
HPyV11 2013 (12) Stool sample N.A N.A 68–70% (160)
29 3.1.4 Human polyomaviruses and associated diseases
Most immunocompetent people with a primary HPyV infection have a nonspecific influenza-like or subclinical course. However, HPyV can replicate and cause specific syndromes in the immunocompromised leading to considerable morbidities. Some HPyV-associated diseases are regularly found in specific patient populations. This suggests that viral determinants interacting within the patient or organs influence the pathological outcome. The reasons for the associations are not clearly understood (179). Different pathologies have been associated with HPyV infections. For instance, BKPyV has been associated with BKPyV-associated nephropathy, however, its association with other diseases such as cancers and autoimmune disease is still been disputed. This presents a hurdle in linking some specific pathologies to HPyVs.
However, a classification of HPyVs-associated pathologies has been described (16, 161)
HPyV12
2013 (13) Liver Colon, rectum,
stool N.A 23–33%
(13, 175) NJPyV/
HPyV13 2014 (14)
Muscle biopsy of pancreatic transplant recipient
Muscle’s endothelia cells and skin
Vasculitic myopathy in pancreatic
transplant recipients (14) N.A
LIPyV
2017 (15) Skin, eye brown
hair N.A NA N.A
30 The notions of HPyVs pathologies
Oncogenic HPyV pathology is characterized by HPyV infecting host cells that allow EVGR expression, but insufficient genome replication and LVGR expression to permit rapid viral release and host cell lysis. This concept has been clearly described in the MCPyV-associated Merkel cell carcinoma, whereby truncation of the LTag and/or Vp1 and MCPyV integration into the host’s genome results in the impairment of MCPyV genome replication and LVGR expression (7). BKPyV LTag uncoupling of host cell activation, LVGR expression and BKPyV release may be associated with urothelial cancers (180).
Cytopathic HPyV pathology is due to rapid viral replication, but with little or no inflammation, resulting to cell lysis. This is seen in JCPyV-associated PML due to an uncontrolled JCPyV replication in the brain of HIV/AIDS patients. The cytopathology is evidence by the loss of the myelin producing cells, leading to neurological deficits and disease progression.
Cytopathic-inflammatory HPyV pathology is characterized by high-level HPyV replication and inflammatory response as a result of cell lysis and necrotic infiltrates of lymphocytes and granulocytes. This pathological pattern is seen in BKPyV- associated PyVAN stage B in kidney allografts.
Immune-reconstituting inflammatory syndrome (IRIS) HPyV pathology is due to high inflammatory response to HPyV antigens, usually during a hasty recovery of the cellular immune response (161, 181). The prototypes are JCPyV-associated PML, which worsen after the commencement of highly active antiretroviral therapy (HAART) (182) or following removal of natalizumab from the plasma in multiple sclerosis patients (183).
Autoimmune HPyV pathology characterized by responding to ’self’ which may be provoked by HPyV antigens. For instance, PyV-LTag interaction with histones and DNA may trigger antibodies against histones or DNA (184-186).
31 Fig. 6: Proposed pattern of HPyV disease during the course of viral replication and immune response adapted from (161). HAART; a colon (:) represents interaction;
BMT, bone marrow transplantation; BKPyVAN-A and BKPyVAN-B, BKPyVAN patterns A and B.
BKPyV, JCPyV, MCPyV, TSPyV and HPyV7 are known to cause diseases in humans (3). Moreover, HPyV6 have been linked to certain diseases (48, 141), while other HPyVs, such as HPyV10 and NJPyV were initially detected from diseased specimens or patients (11, 156) .
3.1.4.1 Human polyomaviruses associated non-cancerous diseases 3.1.4.1.1 BKPyV-associated diseases
Polyomavirus associated nephropathy (PyVAN)
BKPyV was associated with PyVAN in renal transplant recipients. Approximately 1%- 10% of kidney transplant recipients progress to PyVAN and up to 90% of these patients lose their allograft (187-189). PyVAN is a type of acute interstitial nephritis (190) characterized by denudation of the basement membrane (Fig. 7A) and necrosis of the proximal tubules due to BKPyV lytic infection and replication in kidney epithelial cells (191). The introduction of new and more potent immunosuppressive drugs suggests a relationship between immune surveillance disruption and BKPyV
32 reactivation (189, 192, 193). Currently, PyVAN diagnosis includes renal biopsy analyses by immunohistochemistry (IHC) (Fig. 7B) or histopathology to either determine cytopathic effects as a result of lysis of renal tubular epithelia cells. In addition, cytology of urine can be performed to detect decoy epithelial cells, which contain intranuclear viral inclusion bodies and PCR to detect and quantify viral load in urine or blood samples (194, 195). Urine cytology and PCR could be useful for early detection of reactivation and PyVAN (187), thus permitting faster diagnosis and intervention with a consequential increase in graft survival (196).
The risk factor for PyVAN is unclear, however combination of contributing factors from the virus, patient and graft determine the susceptibility to BKPyV. The major risk factor to PyVAN is suggested to be due to an overall degree of immunosuppression and not a particular immunosuppressive treatment (197, 198). More so, the development of PyVAN is not dependent on immunosuppression alone, since the manifestation of the disease is relatively uncommon in non-renal solid organ recipients and other immunosuppressed individuals (161). Furthermore, specific human leukocyte antigen (HLA) mismatches or donor seropositive, older age and male gender are all potential risk factors (187, 199-201).
Until now, there is no specific and effective BKPyV anti-viral treatment available.
Nonetheless, decreasing immunosuppression to permit the immune system to regain control over the infection is the most common first line intervention, although may not be effective in all patients, and may expose the patient to a potential risk of acute graft rejection. Cidofovir, an antiviral nucleoside analogue, inhibits BKPyV DNA replication in vitro (202), but it is nephrotoxic at high doses (203). Studies revealed that at low doses and combined with the reduction of immunosuppression, Cidofovir can be effective in preventing BKPyV reactivation without causing significant nephrotoxicity (204-207). Additionally, the immunosuppressive drug leflunomide has shown some limited successes (207-209). Other antiviral treatments are fluoroquinolone antibiotics and intravenous immunoglobulin (IVIG) (197, 210-212).
More detailed studies are required to evaluate the efficacies of these drugs. Lastly, rapid clearing of the viral source after nephrectomy and then re-transplantation could be another treatment approach.
33
Fig. 7: BKPyV nephropathy (213). (A) Periodic acid Schiff stained section showing tubular epithelia cells with intranuclear viral inclusion bodies (arrows). (B) Immunohistochemistry (IHC) showing a formalin fixed and paraffin embedded tissue section showing BKPyV LTag staining (brown color) with SV40 LTag antibody, which cross reacts with BKPyV LTag.
Polyomavirus-associated hemorrhagic cystitis (PyVHC)
BKPyV is also associated with PyVHC affecting 10%-25% of bone marrow transplant (BMT) patients (214). The disease is probably due to BKPyV reactivation in possibly urothelial cells and renal tubular epithelia cells, resulting in high-level BKPyV replication due to lack of immune surveillance. Furthermore, there is prominent denudation of the cell’s basement membrane, leading to hemorrhage and inflammation (215). The disease is characterized by flank pain, dysuria with or without signs of insufficiency. Additionally, it can be associated with varying grades (grades I-IV) of hematuria (215). A classical BKPyV-associated PyVHC occurs after more than 10 days post-transplantation. PyVHC is not life-threatening, but associated with significant morbidity (216-219). The most commonly applied diagnostic methods for BKPyV-associated PyVHC are PCR detection of viral DNA, IHC to detected LTag expression (Fig. 8A) and urine cytology for the detection of decoy epithelia cells (Fig.
8B) (220). Other sophisticated-diagnostic methods include; magnetic resonance imaging (MRI) or computer tomography (CT) to evaluate the bladder features. The bladder features in infected patients is characterized as; bladder wall thickening, increase mucosa enhancement, mural edema, small bladder capacity and
A B
34 intraluminal clot (221). Like PyVAN, reduction of immunosuppression can be used as a treatment option for PyVHC, whereas exogenous BKPyV-specific T-cell application is being investigated as future therapies (214). Furthermore, antivirals including fluoroquinolone antibiotics, leflunomide, mTOR inhibitors and cidofovir are currently being evaluated for efficacy in treating BKPyV-associated diseases (222-225).
Fig. 8: (A) Bladder biopsy from a patient with PyVHC (www.pathology.mc.duke.edu) depicting denudation and BKPyV infected cells (red arrow head) with enlarged nucleus. (B) PyVHC (220), depicting the characteristic BKPyV-infected cells with decoy cells (arrow) showing enlarged nucleus containing a large intranuclear inclusion BKPyV-associated diseases.
Progressive multifocal leukoencephalopathy (PML)
JCPyV is associated with PML affecting the immunocompromised due to HIV/AIDS, cancer, transplantation and treatment of multiple sclerosis patients with Natalizumab (4, 226-228). A high activity of JCPyV IgG activity is speculative of recent infection with JCPyV, and indicative for an increased risk for PML development in AIDS patients (229). Initially, PML was considered to be a very rare disease associated with immunocompromised patients, usually those with leukemia and lymphomas (93).
With the rise of the HIV/AIDS epidemic, PML resulting from JCPyV infections have drastically risen. Up to 85% of PML cases occur in AIDS patients (230, 231), since not all AIDS-affected patients or even those with very low CD4+ T cells counts come down with PML (232). However, only few sporadic PML cases develop without an underlying immunosuppression (233). In essence, PML is now regarded an AIDS- defining disease (93).
A B
35 PML is clinically characterized by dementia or confusion, speech and vision impairment, varying degree of akinesia or paralysis, in concurrence with immunosuppression and lack of increase in intracranial pressure (94, 233). The progression to PML disease is rapid, with clinically-intensifying symptoms and death within 3-6 months (234), which has however, been improved with the introduction of high-active anti-retroviral therapy (HAART) (235). With respect to the cellular level, PML results as lysis of oligodendrocytes, the myelin producing cells of the brain, leading to lesion in the brain stem, cerebellum and cerebrum and especially along the white and gray matter junction (Fig. 9A) (236, 237). JCPyV DNA sequences have been detected in mononuclear cells and B cells within the brain (238, 239). Enlarged JCPyV Tag-expressing oligodendrocytes with crystalline arrays of viral particles (Fig.
9B) and nuclear inclusion bodies are other features of PML (230, 231). The high frequency of JCPyV-positive PBMCs and widely distributed brain lesions indicate that lymphocytes can serve as a reservoir and a dissemination vehicle (231, 240, 241).
Whether JCPyV infection of the brain is as a result of reactivation of latent infection or de novo invasion of the virus is unclear (81, 242).
The association between PML caused by JCPyV and HIV/AIDS is not clearly understood. However, the link between these two virus-associated disease could be explained by several hypotheses (230, 243, 244). Firstly, HIV establishes a thorough immunosuppression in the host by decreasing JCPyV-specific CD4+ T-cells leading to an uncontrollable JCPyV replication. Specifically, infection of HIV may cause break down of the blood-brain barrier, permitting entry of JCPyV-harboring B cells into the brain. Furthermore, certain cytokines produced as a result of HIV infection could initiate cell signaling events that may activate the JCPyV promoter. Lastly, the HIV Tat protein may activate JCPyV promoters in vitro and increase gene expression (245). Currently, there is no specific antiviral drug to treat PML. However, clinicians now focus on immune reconstitution-based concept.
A B
36 Fig. 8: (A) T2-weighted image of brain from patient with PML (246). The image shows multiple hyper-intense subcortical lesions in the left hemisphere (arrows). (B) Particle of JCPyV in PML lesion (226). Viral particles (VP) scattered all over major areas of the nucleoplasm. Arrows indicate nuclear membrane, while C shows nuclear chromatin.
JCPyV-associated PyVAN
The first case of JCPyV-associated PyVAN was identified in a 15-year old with kidney transplantation (247). Kidney biopsy from the patient stained positive for LTag (Fig. 10) and quantitative PCR revealed high JCPyV load in the urine (247).
Generally, JCPyVAN is found in approximately 1% of kidney transplant patients (248) and about 6% of HIV patients present with JCPyV replication in the kidney (249).
Most patients with JCPyVAN have normal kidney function indicating that JCPyV has a more protracted course or it is apparently less aggressive compared to BKPyVAN.
In addition, BKPyVAN and JCPyVAN have another difference based on the severity of the histological pattern and strong viremia associated with BKPyVAN (187).
Conversely, patients shedding large amounts of JCPyV in urine or patients with parenchyma involvement have been reported to show low viremia levels. This could be due to the basic differences between the biology of JCPyV and BKPyV, which has not been resolved in detail (250, 251). With the debilitating diseases caused by JCPyV and BKPyV, there is an urgent need for the development of specific antiviral therapies.
